Diagnostic elements are important components of clinically relevant analytical methods. This primarily concerns the measurement of analytes such as metabolites or substrates which are determined directly or indirectly for example with the aid of a specific enzyme for the analyte. In this case, the analytes are converted with the aid of an enzyme-coenzyme complex and subsequently quantified. In this process, the analyte to be determined is contacted with a suitable enzyme, a coenzyme and optionally a mediator where the coenzyme is physicochemically changed by the enzymatic reaction. For example, the coenzyme may be oxidized or reduced by the enzymatic reaction. If a mediator is used, it typically transfers the electrons released from the reduced coenzyme during the conversion of the analyte onto an optical indicator or the conductive components of an electrode so that the process can be detected photometrically or electrochemically. A calibration yields a direct relationship between the measured value and the concentration of the analyte to be determined.
Diagnostic elements known from the prior art are characterized by a limited shelf-life and by special requirements, such as cooling or dryness, for the environment in which they are stored in order to achieve this shelf-life. With certain types of application, for example in the case of tests that are carried out by the end user himself such as blood glucose self monitoring, erroneous results that can hardly be detected by the user may therefore occur due to a false unnoticed incorrect storage of the measuring system and may lead to improper treatment of the respective disease.
The erroneous results are primarily based on the fact that the enzymes, coenzymes and/or mediators used in such diagnostic elements may generally react sensitively to moisture and heat and, as a result, are inactivated in whole or in part. Thus, when glucose is detected by means of a glucose dehydrogenase/NAD system under warm and humid environmental conditions for example, the activity of the glucose dehydrogenase enzyme as well as the content of the NAD coenzyme decrease over time and the decrease in the coenzyme generally proceeds considerably more rapidly than the loss of activity of the enzyme.
A known measure that is used to increase the stability of diagnostic elements is to use stable enzymes, such as those enzymes from thermophilic organisms. Furthermore, it is possible to stabilize enzymes by chemical modification such as cross-linking, or by mutagenesis. Moreover, enzyme stabilizers such as trehalose, polyvinyl pyrrolidone and serum albumin for example can be added or the enzymes can be enclosed in polymer networks by, for example, photopolymerization.
Another method for improving the stability of diagnostic elements is to use stabilized coenzymes. Whereas native coenzymes such as NAD and NADP or their reduced forms NADH and NADPH for example are relatively unstable under basic or acidic conditions due to the lability of the glycosyl bond between the ribose and the pyridine unit, various derivatives of NAD/NADH and NADP/NADPH have been described in the literature in recent years which significantly increase the stability of the coenzyme by modification of the nicotinamide group or the ribose unit.
The stability of diagnostic elements can also be increased by using stable mediators. Thus, the specificity of tests is increased and interferences during the reaction are eliminated by using mediators having a redox potential that is as low as possible. However, the redox potentials of the enzyme/coenzyme complexes form a lower limit for the redox potential of mediators. If the potential is lower than this limit, the reaction with the mediators is slowed down or even stopped.
However, the components of chemical detection reagents that are used in diagnostic elements do not all have the same stability in practice, but rather are subject to decomposition processes that occur at different rates. Thus, when carbaNAD is used as a coenzyme for example, the effect occurs that the coenzyme remains very stable over a long period even under humid conditions and at elevated temperatures due to the carbacyclic sugar unit, whereas the activity of a native enzyme used in the diagnostic element continuously decreases. Likewise, if a combination of a stabilized enzyme and a native coenzyme is used for example, the activity of the enzyme can be maintained over a long period whereas the amount of coenzyme rapidly decreases to a greater or lesser extent due to thermal and/or hydrolytic decomposition.
In order to avoid erroneous results when determining analytes, diagnostic elements should allow a determination of whether the individual components of a detection reagent used in the diagnostic element are still in a functional form at the time of measurement and that the detection reagent is inasmuch suitable for the qualitative and/or quantitative detection of the analyte. In this connection, the determination of the enzyme activity is particularly problematic because the inactivation of an enzyme is firstly caused by a change in the conformation of the protein and hence no optically-active or electrochemically-active particles are formed because there is no change in the constitution.
In view of the foregoing, one non-limiting object of the present application is to provide a stable diagnostic element for determining an analyte such as glucose in which the disadvantages of the prior art are at least partially eliminated. In one aspect, the diagnostic element should ensure that in the case of a greatly reduced or completely absent functionality of individual components of a detection reagent, one avoids showing measurement results that are allegedly correct with respect to a particular analyte.
In one non-limiting aspect, the above-identified object is achieved by a diagnostic element for determining at least one analyte that includes a detection reagent that is specific for the analyte and an indicator analyte having a defined amount of the at least one analyte. The indicator analyte is present in a releasable form that is inaccessible for a reaction with the detection reagent.